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International Journal of Bioprinting 3D-bioprinted hydrogel for pulp regeneration

gene GAPDH was used as the endogenous control. Primer sequences used for qPCR are listed in Table S1
Changes in gene expression were measured using the 2 -ΔΔCT (Supplementary File).
method, and the final results were presented as fold change
relative to the two-dimensional (2D) group. The primer 2.10. Cell viability assay
sequences used are listed in Table S1 (Supplementary File). The viability of encapsulated cells in the 3D-bioprinted
For validation, the experiment was repeated for at least hydrogel constructs in vitro was examined by Live/Dead
26
three times. assay. Briefly, the hDPSCs-laden DPGC or bulk hydrogel
constructs were cultured in 6-well plates (Corning Inc.,
2.8. Immunofluorescent staining of YAP Kennebunk, ME, USA) and maintained in full culture
Immunofluorescent staining of YAP was performed for medium for different durations (1, 5, and 7 days). Before the
the hDPSCs encapsulated in the 3D-bioprinted constructs examination, the constructs were immersed in the calcein
after 3 days of culture. The primary antibodies against YAP acetoxymethyl ester (calcein-AM) and propidium iodide
(1:100 dilutions; GeneTex, Irvine, CA, USA) were used, and (PI) reagent following the manufacturer’s instructions, and
the fluorescent signal of YAP was detected using CLSM. were visualized using fluorescent microscopy.
2.9. Evaluation of odontogenesis The viability of cells in the constructs was examined on
a mouse subcutaneous implantation model. hDPSCs were
2.9.1. Odontogenic differentiation encapsulated in the 3D-bioprinted DPGC or bulk hydrogel
For the odontoblastic differentiation assay, hDPSCs were constructs and implanted subcutaneously into 3-week-
separately encapsulated in 3D-bioprinted DPGC and bulk old immunodeficient mice. The samples were harvested
hydrogel constructs in 12-well plates, and directly seeded on days 7 and 14 after implantation. Then, these samples
on the TCPs. The hDPSCs-laden constructs were first were immediately fixed in 4% PFA for 24 h, embedded in
cultured in a complete medium for 24 h for cell attachment. paraffin blocks, and sectioned at a thickness of 5–10 μm.
Afterward, the constructs were treated with odontogenic Paraffin tissue sections were deparaffinized in xylene and
media (Cyagen Biosciences, Guangzhou, China) for 7 and rehydrated through graded ethanol solutions. Finally,
14 days. the sections were stained with hematoxylin–eosin (H&E;
Sigma Aldrich, St. Louis, MO, USA) according to the
2.9.2. Alkaline phosphatase (ALP) activity manufacturer’s instructions.
Alkaline phosphatase (ALP) activity was detected using
the Alkaline Phosphatase Assay Kit (Beyotime Institute 2.11. Cell proliferation assay
of Biotechnology, Jiangsu, China) according to the The proliferation of hDPSCs encapsulated in the
manufacturer’s protocol. Briefly, the 3D-bioprinted cell- 3D-bioprinted hydrogel constructs was examined using
laden hydrogel constructs were homogenized in GelMA Cell Counting Kit-8 according to the manufacturer’s
lysis buffer (EFL) at 37°C for 30 min. Lysate was centrifuged instructions. Briefly, the 3D-bioprinted hDPSCs-laden
at 1000 rpm at room temperature for 5 min. After removal DPGC or bulk constructs were cultured in 12-well plates
of the supernatants, the deposits were lysed with RIPA and maintained in complete culture medium for different
buffer without the inhibitors (Beyotime Institute of durations (1, 5, and 7 days). Before examination, the
Biotechnology) on ice for 1 h. Lysates were centrifuged at hDPSCs-laden constructs were incubated with CCK-
1000 rpm at 4°C for 5 min. The supernatant was obtained 8 reagent at 37°C for 2.5 h. The absorbance at 405 nm
for ALP assay using p-nitrophenyl phosphate (p-NPP) as a was detected with the spectrophotometer (BioTek
phosphatase substrate and alkaline phosphatase standard Instrumentals, Winooski, VT, USA) on days 1, 5, and 7
supplied with the kit. The absorbance was measured at 405 after culture.
nm, and the amount of ALP in the cells was normalized
against total protein content. The total protein amount 2.12. Cell migration assay
was quantified using an Enhanced BCA Protein Assay Kit Cell migration assay was performed in a 6.5 mm transwell
(Beyotime Institute of Biotechnology) in adherence with with 8.0 µm pore polycarbonate membrane inserts in a 24-
the manufacturer’s instructions. well format (Corning Inc.). The 3D-bioprinted hDPSCs-
laden DPGC or bulk constructs were placed into the upper
2.9.3. Gene expression analysis chambers of transwell plates and cultured in serum-free
After 7 and 14 days of culture, qPCR was performed to medium. The lower chambers were filled with 10% FBS.
measure the expression of dentin-related genes, such The samples were incubated at 37°C for 7 days, and then
as ALP, RUNX2, OCN, OPN, and DSPP, for hDPSCs washed with PBS. After fixation with 4% PFA and staining
in different groups based on the protocols provided in with crystal violet (BBI, Shanghai, China), hDPSCs of
section 2.7. The experiments were repeated 3 times. each filter were counted in five random regions from three

Volume 10 Issue 3 (2024) 304 doi: 10.36922/ijb.1790